Synthetic lethality between ATR and POLA1 reveals a potential new target for individualized cancer therapy

The ATR-CHK1 pathway plays a fundamental role in the DNA damage response and is therefore an attractive target in cancer therapy. The antitumorous effect of ATR inhibitors is at least partly caused by synthetic lethality between ATR and various DNA repair genes. In previous studies, we have identified members of the B-family DNA polymerases as potential lethal partner for ATR, i.e. POLD1 and PRIM1. In this study, we validated and characterized the synthetic lethality between ATR and POLA1. First, we applied a model of ATR-deficient DLD-1 human colorectal cancer cells to confirm synthetic lethality by using chemical POLA1 inhibition. Analyzing cell cycle and apoptotic markers via FACS and Western blotting, we were able to show that apoptosis and S phase arrest contributed to the increased sensitivity of ATR-deficient cancer cells towards POLA1 inhibitors. Importantly, siRNA-mediated POLA1 depletion in ATR-deficient cells caused similar effects in regard to impaired cell viability and cumulation of apoptotic markers, thus excluding toxic effects of chemical POLA1 inhibition. Conversely, we demonstrated that siRNA-mediated POLA1 depletion sensitized several cancer cell lines towards chemical inhibition of ATR and its main effector kinase CHK1. In conclusion, the synthetic lethality between ATR/CHK1 and POLA1 might represent a novel and promising approach for individualized cancer therapy: First, alterations of POLA1 could serve as a screening parameter for increased sensitivity towards ATR and CHK1 inhibitors. Second, alterations in the ATR-CHK1 pathway might predict in increased sensitivity towards POLA1 inhibitors.


Introduction
Cancer therapy is one of the most permanent changing subjects of modern medicine.Currently, a main goal is to establish more efficient cancer treatments by using individualized and targeted approaches for tumor cells and thereby to minimize the frequency and severity of side effects.To this end, genotype-based cancer treatment represents a promising tool.During carcinogenesis the accumulation of mutationsparticularly within the DNA repair pathwaysis one crucial process for the malignant transformation of normal cells to cancer cells.To compensate the impairment in certain DNA repair pathways due to the accumulation of mutations, other DNA repair pathways are often upregulated in cancer cells [20].This process facilitates novel therapeutic approaches through practical application of the principle of synthetic lethality.
Synthetic lethality describes the relationship between two genes where inactivation of either gene alone has no effect, but inactivation of both genes is lethal to the cell carrying these mutations [32].Thus, by pharmacologically targeting a gene, which compensates the lossmediated by mutations during carcinogenesisof its synthetically lethal Abbreviations: ATR, ataxia telangiectasia and Rad3-related protein; ATRIP, ATR interacting protein; CHK1, Checkpoint kinase 1; COSMIC, Catalogue of Somatic Mutations In Cancer and Cell; IC50, half-maximal inhibitory concentration; MMC, Mitomycin C; NTC, non-transfected cells; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; RPA, replication protein A; ssDNA, single stranded DNA.partner, synthetic lethality increases toxicity specifically towards cancer cells.A well-known example with clinical impact is the role of poly (ADP-ribose) polymerase (PARP) inhibitors in tumors harboring BRCA1/2 mutations [15].This targeted approach is already used for multiple cancer entities including breast, ovarian and pancreatic cancers [26].
In our previous studies on the synthetically lethal relationship between ATR and POLD1 and ATR and PRIM1 [21,23,24], preliminary experiments indicated another synthetically lethal relationship, namely between ATR and POLA1, encoding the catalytic subunit of the DNA polymerase α [8].This assumption was further supported by Rogers et.
al. [35], which also confirmed a synthetic lethal relationship between CHK1 and multiple DNA polymerases such as POLA1, POLE and POLE2.
To gain a more detailed understanding of the effects already described and thereby provide a basis for clinical benefit, we have investigated and characterized this relationship between ATR and POLA1.First, we applied a well-described cellular model of ATRdeficiency in the human colorectal cancer cell line DLD-1 [16,21,22,46], consisting of parental DLD-1 cells (subsequently referred to as ATR +/+ cells) and a derived DLD-1 cell clone homozygously harboring the a hypomorphic ATR Seckel mutation (subsequently referred to as ATR s/s cells).While total loss of ATR is incompatible with cell viability [5], the Seckel mutation applied in this system causes subtotal ATR protein depletion without any detectable effect on viability.To model POLA1 deficiency, we applied ST1926, an atypical retinoid that has already been shown to inhibit POLA1 and proliferation of colorectal cancer cells [1].
Vice versa, as POLA1 mutations occur in about 7 % of colorectal and pancreatic cancer samples, we additionally used four different ATR and CHK1 inhibitors to assess the chemical inducibility of synthetic lethality in various POLA1-depleted colon and pancreatic cancer cell lines.
Our results illustrate a novel potential approach for individualized cancer therapy using specific ATR/CHK1 inhibitors or POLA1 inhibitors for the individualized treatment of ATR-or POLA1-deficient tumors, respectively.

Transfection
Protein knockdown was achieved by transfection experiments.For this purpose, HiPerFect (QIAGEN, Hilden, Germany) was mixed with 5 nM siRNA targeting POLA1 (ATGGCAGTCTTTCCTCTCTTA) (QIAGEN, Hilden, Germany) in medium without FBS.After 20 minutes of incubation, this mixture was added to recently plated cells.Allstars negative (non-targeting) and medium without FBS were established as controls to monitor off-target effects.

Cell proliferations assays
Drug sensitivity was assessed by cell proliferation assays in ATR +/+ , ATR +/s and ATR s/s cells.Therefore, 1500 cells of ATR +/+ , ATR +/s and ATR s/s were sown in 96-well plates.After 24 h, they were exposed to the above-mentioned drugs for 120 h.0.2 % SYBR-green (Lonza, Cologne, Germany) solution in Aqua destillata was added and fluorescence was measured by Victor 3 V plate reader (PerkinElmer, Waltham, MA).Survival fraction was determined by dividing treated cells by an untreated control group.

Cell viability assays
Cell viability assays examined either the impact of POLA1 depletion through transfection or the sensibility towards various drugs.For the former, 12,500 -50,000 ATR +/+ cells and 15,000 -60,000 ATR s/s cells were transferred in 6-well plates, transfected as described above and incubated for 72 -168 h.For the latter, 140,000 ATR +/+ , RKO, HCT-116 or PaTu8988t cells were transferred in 6-well plates and transfected as described above.After 72 h, 2,000 -25,000 cells of POLA1 depleted cells and control samples were plated in 96-well plates.After another 24 h, they were treated with various drugs for 120 h.
Afterwards, cells were washed with Dulbecco´s Phosphate Buffered Saline (DPBS) (Thermo Fisher Scientific, Schwerte, Germany) and treated with MTT.For the MTT-reagent, 0.5 % Thiazolylblue (Roth, Karlsruhe, Germany) solution in DPBS was added to RPMI without FBS.This reagent was added to each well and incubated for 90 minutes.Absorption was measured using a Multiscan FC (Thermo Fisher Scientific, Schwerte, Germany), and cell viability was determined by dividing treated cells by an untreated control group.

Flow cytometry
To investigate cell cycle and apoptosis, flow cytometry was performed according to the methods previously described by our group [23,24].62,500 -250,000 ATR +/+ cells and 75,000 -300,000 ATR s/s cells were seeded in 6-well plates and were then treated with ST1926 24 h after seeding.Cells were harvested after a subsequent 24, 48 and 72 h.For cell cycle analysis, cells were washed and stained with propidium iodide (PI; 0.1 % sodium citrate, 0.1 % Triton X-100, 50 μg/ml propidium iodide) according to previously described protocols [31].For apoptosis analysis, cells were washed and stained with FITC-conjugated Annexin-V (Biolegend, San Diego, CA) diluted 1:40 in Hanks Balanced Salt solution (HBSS) (Thermo Fisher Scientific, Schwerte, Germany) for 20 minutes at room temperature in the dark.Afterwards, 1 µl propium iodide (1mg/ml, Thermo Fisher Scientific, Schwerte, Germany) was immediately added before measurement.
The BD FACSCanto II from BD Biosciences (San Jose, CA) and the FlowJo v10 software from FlowJo, LLC (Ashland, OR) were used to analyze both cell cycle and apoptosis.For each sample, a minimum of 30,000 gated events were assessed.According to Wlodjowic et al. [47] PI − /Annexin V + were interpreted as early apoptotic and PI + /Annexin V + cells as late apoptotic cells.

Statistical analyses
GraphPad Prism 9.5.0 (La Jolla, CA, USA) was used for all statistical analyses.Error bars represent ± SD.A minimum of three separately experiments were performed.Statistical analysis was performed by twoway ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.

Synthetic lethality between ATR and POLA1 in an ATR-knock-in DLD-1 model
To verify the synthetic lethal interaction between ATR and POLA1 we initially applied a well-described model of ATR-deficient DLD-1 human colorectal cancer cells [16,21,22,46].First, we confirmed and quantified ATR and POLA1 expression in these cell lines via Western blotting (Fig. 1A and B).In concordance with our previous studies, ATR +/+ cells showed the highest ATR protein levels, whereas the ATR levels slightly decreased in ATR +/s cells and were almost undetectable in ATR s/s cells (Fig. 1A).Interestingly, POLA1 protein levels reciprocally increased with decreasing ATR protein levels, showing the highest expression in ATR s/s cells and the lowest expression in ATR +/+ cells (Fig. 1B).Next, we confirmed the previously described increased sensitivity of ATR s/s cells towards the DNA interstrand-crosslinking agent MMC [17] as compared to ATR +/+ and ATR +/s cells (Fig. 1C).Then, we assessed the sensitivity of ATR s/s as compared to ATR +/+ cells towards chemical POLA1 inhibition using ST1926 (Fig. 1D).Notably, with IC 50 ratios of 6 and 4, respectively, ST1926 showed at least comparable if not even stronger effects than MMC.Taken together, these data illustrate a synthetically lethal relationship between ATR and POLA1 through simultaneous impairment of ATR and POLA1 in DLD-1 colorectal cancer cells.Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.Asterisks were used for comparisons between ATR +/+ and ATR s/s , triangles for comparisons between ATR +/+ and ATR +/s , hash symbols for comparisons between ATR +/s and ATR s/s .Western blots show representative results of at least three separately performed experiments.

ST1926-mediated effects on cell cycle distribution in ATR +/+ cells vs ATR s/s cells
To assess whether cell cycle arrest or apoptosis contributed to the increased sensitivity of ATR s/s cells towards ST1926, we next examined the cell cycle distribution including the sub-G 1 fraction in ATR +/+ cells and ATR s/s cells 24 h, 48 h and 72 h after ST1926 treatment.Upon treatment with ST1926, both ATR +/+ and ATR s/s cells showed increased S phase-and decreased G 2 /M phase-fractions (Fig. 2A).In contrast, only ATR s/s cells but not ATR +/+ cells showed a significantly increased sub-G 1 fraction upon ST1926 treatment (Fig. 2B).This effect increased in a time-dependent manner with approximately 10 % of cells in sub-G 1 at 72 h post treatment (Fig. 2C).Thus, the POLA1 inhibitor ST1926 induced S phase-arrest in both ATR-proficient and ATR-deficient DLD-1 cells, but consecutive apoptosis (as indicated by the subG 1 fraction) only and specifically in cells lacking ATR.

ST1926-mediated induction of apoptosis in ATR +/+ cells vs ATR s/s cells
To evaluate whether the observed increased sub-G 1 fraction in ATR s/s cells was indeed ascribable to apoptosis, we next performed Annexin V assays in ATR +/+ cells and ATR s/s cells upon ST1926 treatment.While neither ATR +/+ cells nor ATR s/s cells showed an increase of Annexin V + without ST1926 treatment (data not shown), we observed an increase of early (PI − /Annexin V + ) as well as late apoptosis (PI + /Annexin V + ) in ATR s/s cells as compared to ATR +/+ cells upon ST1926 treatment (Fig. 3A and B).While this effect was not yet visible after 24 h, the percentage of Annexin V + ATR s/s cells increased continuously over the following time points.To characterize apoptosis more specifically and mechanistically, we examined the protein levels of essential apoptosis markers via Western blotting in ATR s/s and ATR +/+ cells 24 h, 48 h and 72 h post treatment with ST1926 (Fig. 3C).We found an ST1926induced increase of PARP cleavage exclusively in ATR s/s cells but not in ATR +/+ cells.Accordingly, the cleavage of caspase 3, the main effector caspase in the execution phase of apoptosis [13], was also increased specifically in ATR s/s cells at 48 h and 72 h but not in ATR +/+ cells after ST1926 treatment.Taken together, the increased sensitivity of ATR s/s cells towards chemical POLA1 inhibition can therefore be explained, at least in part, by S-phase arrest and subsequent apoptosis as contributing mechanisms.siRNA-mediated POLA1 depletion in ATR +/+ cells vs ATR s/s cells We wanted to examine the effects of a simultaneous inactivation of ATR and POLA1 in another, perhaps more specific model of POLA1 inactivation.Therefore, we tested the effects of POLA1 protein depletion in ATR +/+ cells vs ATR s/s cells.Efficient POLA1 depletion was confirmed in both ATR +/+ cells and ATR s/s cells at 72 h, 96 h and 120 h after siPOLA1 transfection (Fig. 4A).We observed an increased sensitivity of ATR s/s cells 72 -168 h after siPOLA1 transfection as compared to ATR +/ + cells (Fig. 4B, C).To test whether apoptosis contributed to these effects as similarly shown for ST1926 -we further quantified protein levels of essential apoptosis markers via Western blotting in ATR +/+ cells vs ATR s/s cells upon siPOLA1 transfection.Increased levels of cleaved PARP, pChk1 and cleaved Caspase 3 was observed at 96 h and 120 h after siPOLA1 transfection specifically in ATR s/s cells (Fig. 4D).After siRNA-mediated POLA1 depletion, only the ATR s/s cells showed increased cleaved caspase 3 levels.Similarly, the levels of Poly(ADPribose) polymerase (PARP) were elevated in POLA1 depleted ATR s/s cells.In addition, CHK1, the major downstream effector kinase of ATR [34,49] was phosphorylated after POLA1 depletion only in ATR s/s .These effects became apparent at 96 h and lasted until 120 h after transfection.Thus, siRNA-mediated POLA1 depletion induced similar effects as did ST1926 in ATR-deficient cells, further supporting our hypothesis of synthetic lethality between ATR and POLA1 in colorectal cancer cells.

siRNA-mediated POLA1 depletion sensitizes HCT-116 and PaTu8988t cells towards ATR and CHK1 inhibitors
To test whether our data initially obtained in DLD-1 were generalizable beyond one cell line or tumor entity, we next assessed whether POLA1 depletion also sensitized other cancer cell lines towards ATR/ CHK1-inhibition.Therefore, the colorectal cancer cell line HCT-116 and the pancreatic cancer cell line PaTu8988t were treated with ATR and CHK1 inhibitors, respectively.After verification of efficient POLA1 depletion (Fig. 6A and B), we were able to show a significant increased sensitivity towards treatment with ATR inhibitors AZD6738 and VE-822 upon siPOLA1-transfection in both cell lines (IC 50 ratios between 3 and 7; Fig. 6C).Similarly, we observed in both cell lines a significant increased sensitivity towards treatment with CHK1 inhibitors LY2603618 and MK-8776 upon siPOLA1-transfection (IC 50 ratios between 3 and 13; Fig. 6D).Thus, siPOLA1-mediated sensitization to ATR and CHK1 inhibitors is not a cell line-specific phenomenon of DLD-1 cells but can be generalized to a panel of cell lines of different tumor entities.

Discussion
Using siRNA-library screening of DNA repair genes, we previously identified several genes from B-family DNA-polymerases as potential synthetic lethal partners for the checkpoint kinase ATR of which we consecutively characterized POLD1 and PRIM1 in follow-up studies [21,23,24].In this study, we validated and characterized the relationship between ATR and another previously identified candidate partner, i.e.POLA1.To this end, we applied various genetic, epigenetic and chemical experimental settings to model ATR-and POLA1-impairment.
In line with our initial hypothesis of synthetic lethality between POLA1 and ATR, we first demonstrated in ATR +/+ and ATR s/s cells that POLA1 protein levels increased reciprocally with decreasing ATR protein levels, suggesting a perhaps compensatory activation of POLA1 upon inactivation of ATR.This hypothesis was further corroborated by the increased sensitivity of ATR-deficient DLD-1 cells towards treatment with the POLA1 inhibitor ST1926.
To gain more mechanistic insights into the synthetically lethal relationship between ATR and POLA1, we next investigated whether cell cycle perturbations or apoptosis contributed to the observed effects.ST1926 has already been reported to induce cytostatic and cytotoxic effects including S-phase arrest and apoptosis [1][2][3]43].Besides, apoptosis and cell cycle arrest has also been reported in glioblastoma cell lines treated with ST1926.However, compared to the previously described S phase arrest, this was associated with G0/G1 arrest and a significant reduction of cells in S phase [12].Interestingly, in our study both ATR-proficient and ATR-deficient cells displayed an increased S and decreased G 2 /M phase upon ST1926 treatment, while increased subG 1 phase was only detectable in ATR-deficient cells.This could indicate that after ST1926-induced cell cycle arrest only ATR-proficient cells can initiate sufficient DNA damage repair to prevent replication catastrophe, while ATR-deficient cells cannot [41].
ATR is activated by RPA coated ssDNA and suppresses the emergence of more ssDNA and the RPA exhaustion by inhibiting origin firing.Furthermore, it is proficient to prevent replication catastrophe [42].Interestingly, Ercilla et.al. [14] showed that POLA1 activity is also capable to prevent replication catastrophe by avoiding accumulation of ssDNA and RPA exhaustion.Thus, we presume that vice versa the inhibition of POLA1 induces accumulation of ssDNA and consumption of RPA.This assumption is supported by RPA phosphorylation after sole POLA1 depletion, indicating that lower levels of POLA1 might correlate with the amount of replicative stress [35].If POLA1 is thus inhibited in ATR-or CHK1-deficient cancer cells, global RPA exhaustion and therefore replication catastrophe culminating into apoptosis might be the consequence.
As we observed an increased subG 1 fraction in ATR-deficient cells upon POLA1 inhibition, we assumed that apoptosis contributed to the synthetically lethal interactions between ATR and POLA1, which was confirmed by the elevated levels of Annexin V, Caspase 3 cleavage and PARP cleavage in ATR and POLA1 co-depleted cells.Caspase 3 is a central executioner of regulated cell death and is activated by other caspases [18].PARP cleavage itself is catalyzed by Caspase 3 to prevent depletion of PARP substrates and is therefore also an indicator for apoptosis [4,38].Thus, the growth inhibition and decreased cell viability induced by the simultaneous ATR and POLA1-impairment can be ascribed at least partially to apoptosis.
Further, POLA1 depletion combined with ATR/CHK1 deficiency induced by chemical ATR/CHK1 inhibition decreased cell viability significantly in our experiments.This phenomenon was observed not only in our initial model of DLD-1 colorectal cancer cells, but was generalizable to various cell lines of different tumor entities.Although POLA1 knockdown was also successfully performed in the colorectal cancer cell line RKO, we did not observe similar effects upon treatment with ATR or CHK1 inhibitors, respectively, in POLA1 depleted cells (data not shown), indicating that expectedly, the therapeutic targeting of POLA1 impaired or vice versa ATR impaired cells using the respective inhibitors will not unequivocally work in all cell lines.This is likely explainable by the highly heterogenous mutation patterns of different tumor cell lines, some of which could harbor mutations that overrule the synthetically lethal effects between ATR/CHK1 and POLA1.
Of note, our data demonstrating increased sensitivity of ATR/CHK1 inhibitors in POLA1 depleted cells are strongly supported by already published data [35] showing synthetic lethality between CHK1 and B-family DNA polymerase including POLA1 in both lung and colorectal cancer cells.In that study, it was demonstrated that the simultaneously chemical inhibition of POLA1 and CHK1 increases replication stress, DNA damage and apoptosis compared to single drug using.Some of these B-family DNA polymerase members, e.g.POLD1 and POLE, are already known to be mutated in some familial colorectal carcinomas and adenomas [33].Although such a correlation is not yet known for POLA1, POLA1 mutations are found in 6,7 % of tested colon cancer samples and in 22 out of 55 tested colon cancer cell lines according to Catalogue of Somatic Mutations In Cancer and Cell (COSMIC) [10] by Sanger Institute.In addition, studies showed that colorectal cancer cells which are resistant towards ST1926 and its parent molecule CD437 develop POLA1 mutations [1,19].Future studies are needed to determine, whether these previously described POLA1 mutations in other malignant entities are similarly interacting synthetically lethal with ATR and CHK1 inhibitors as has been shown in our study.
The potential impact on clinical anticancer therapy might not only be restricted to pathogenic POLA1 mutations but potentially also extendable to mere altered POLA1 expression, as low POLA1 expression leads to increased sensitivity against CHK1 inhibitors [35], consistent with high yH2AX levels as an indicator for increased replication stress in CHK1 and POLA1 siRNA-mediated co-depleted cells [40].Therefore, altered POLA1 expression could potentially also serve as indicator for tumor response after treatment with CHK1 inhibitors.
In addition, Takahashi et al. [39] developed a transcriptional profile, the repstress score, for replication stress that includes POLA1 as a marker.The repstress score has been shown to predict sensitivity to ATR inhibitors and is therefore a potential tool for patient selection.Although many other factors are included in this score, it further corroborates the relationship between POLA1 and ATR as characterized in our study.
Finally, CHK1 frameshift mutations might contribute to tumorgenesis in microsatellite instable colon carcinomas due to consecutive defects in the DNA damage response [27].Although COSMIC by Sanger Institute only counted approximately 2 % of CHK1 mutations in the large intestine, many of these alterations could have a pathological impact with almost 30 % frameshift insertions or deletions [10].Therefore, it would be interesting to analyze the effects of POLA1 inhibitors specifically in cancer cells harboring these mutations.Unfortunately, a Phase I clinical trial in patients with advanced ovarian cancer displayed reduced bioavailability of ST1926 due to glucuroconjugation [36] and was therefore not further investigated.Nevertheless, ST1926 was able to reduce tumor burden and prolong survival in a murine model.
In conclusion, our study suggests that the synthetically lethal effects of simultaneous impairment of ATR and POLA1 in cancer cells might represent a novel and promising approach for individualized cancer therapy.Specific functional POLA1 mutations could potentially serve as a new biomarker for selection of tumors with an increased sensitivity towards ATR or CHK1 inhibitors and, vice versa, CHK1 mutations might serve as a new biomarker to predict an increased sensitivity towards POLA1 inhibitors.Currently, novel compounds with POLA1 inhibitory activity are tested in murine xenograft models [7].Interestingly, two novel dual inhibitors, MIR002 and GEM144, have been shown to specifically inhibit POLA1, causing S-phase arrest and activation of the ATR pathway, and have also demonstrated significant antitumor activity when administered orally in two different human orthotopic malignant pleural mesothelioma xenografts [11].Moreover, many chemical ATR and CHK1 inhibitors are already investigated in phase I or II clinical  trials (e.g. the ATR inhibitor AZD6738 [29,45] and M6620 [48] or CHK1 inhibitors MK-8776 [44], LY2603618 [37] and SRA737 [25,28]).These studies, especially when stratifiable by mutational tumor status in regard to POLA1, ATR and CHK1, respectively, will further elucidate the potential clinical implications of our study.
H.E. Schneider et al.manufacturer's instructions.ß-Actin was included as a loading control.All western blots are representative of at least three separate experiments.

Fig. 1 .
Fig. 1.Synthetic lethality between ATR and POLA1 in an ATR-knock-in DLD-1 model.(A) Confirmation of ATR expression in ATR +/+ , ATR +/s and ATR s/s cells via Western blotting.(B) Confirmation of POLA1 expression in ATR +/+ , ATR +/s and ATR s/s cells via Western blotting.(C) Assessment of MMC sensitivity on the proliferation of ATR +/+ , ATR +/s and ATR s/s cells via proliferation assay 120 h post treatment.(D) Assessment of ST1926 sensitivity on the proliferation of ATR +/+ and ATR s/s cells via MTT-assay 120 h post treatment.Data points are based on triplicate wells of a minimum of three separate experiments.Error bars represent ± SD.Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.Asterisks were used for comparisons between ATR +/+ and ATR s/s , triangles for comparisons between ATR +/+ and ATR +/s , hash symbols for comparisons between ATR +/s and ATR s/s .Western blots show representative results of at least three separately performed experiments.

Fig. 2 .
Fig. 2. ST1926-mediated effects on cell cycle distribution in ATR +/+ cells vs ATR s/s cells.(A) Cell cycle profile of ATR +/+ and ATR s/s cells 24 h, 48 h and 72 h after ST1926 treatment assessed by FACS analysis.(B) Histograms of ATR +/+ and ATR s/s cells 48 h after ST1926 [40nM] treatment as measured by FACS analysis.(C) SubG 1 fraction of the cell cycle analysis (Fig. 2A) as measured by FACS analysis.Data points are based on triplicate wells of a minimum of three separate experiments.Error bars represent ±SD.Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.

Fig. 3 .
Fig. 3. ST1926-mediated induction of apoptosis in ATR +/+ cells vs ATR s/s cells.(A) Quantification of early apoptotic (PI − /annexin V + ) and late apoptotic cells (PI + / annexin V + ) in ATR +/+ and ATR s/s cells at 24, 48 and 72 h post treatment with 100 nM ST1926 as measured by flow cytometry.(B) Representative histograms of data shown in (A) of ATR +/+ and ATR s/s cells 72 h post treatment with 100 nM ST1926 as measured by flow cytometry.(C) Representative results of essential apoptotic markers in ATR +/+ and ATR s/s cells by Western blotting 24, 48 and 72 h post treatment with 100 nM ST1926.Data points are based on triplicate wells of a minimum of three separate experiments.Error bars represent ± SD.Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.

Fig. 4 .
Fig. 4. siRNA-mediated POLA1 depletion in ATR +/+ cells vs ATR s/s cells.(A) Depletion of POLA1 in ATR +/+ and ATR s/s cells 72, 96 and 120 h after siRNA mediated knock-down shown via Western blotting.A minimum of three independent experiments were performed.(B) Cell viability after siRNA mediated knock-down in ATR +/+ and ATR s/s cells 72, 96 and 120 h after transfection.(C) Cell viability after siRNA mediated knock-down in ATR +/+ and ATR s/s cells 144 and 168 h after transfection.Cell viability of siPOLA1-and mock-transfected cells was calculated on the basis of non-treated cells.Data points are based on triplicate wells of a minimum of three separate experiments.Error bars represent ±SD.Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.(D) Protein quantification of apoptotic markers in ATR +/+ and ATR s/s cells at 72, 96 and 120 h after siRNA mediated POLA1 knock-down via Western blotting.A minimum of three independent experiments were performed.

Fig. 5 .
Fig. 5. siRNA-mediated POLA1 depletion sensitizes ATR-proficient DLD-1 cells towards ATR and CHK1 inhibitors.Sensitization towards (A) ATR inhibitors and (B) CHK1 inhibitors was measured after 120 h of drug treatment in POLA1-depleted ATR +/+ cells vs. control and mock-transfected ATR +/+ cells by MTT-assay.Data points are based on triplicate wells from a minimum of three separate experiments.Error bars represent ± SD. performed.Statistical analysis was performed by twoway ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.Asterisks were used for comparisons between NTC and siPOLA1, triangles for comparisons between NTC and mock, hash symbols for comparisons between mock and siPOLA1.

Fig. 6 .
Fig. 6. siRNA-mediated POLA1 depletion sensitizes HCT-116 and PaTu8988t cells towards ATR and CHK1 inhibitors.POLA1 depletion after siRNA mediated knockdown in (A) HCT-116 cells and (B) PaTu 8988t cells 72 h after transfection via Western blotting.A minimum of three independent experiments were performed.Sensitization towards (C) ATR inhibitors and (D) CHK1 inhibitors was measured after 120 h of drug treatment in POLA1 depleted HCT-116 or PaTu 8988t cells vs. control and mock-transfected HCT-116 or PaTu 8988t cells by MTT-assay.Data points are based on triplicate wells from a minimum of three separate experiments.Error bars represent ± SD.Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test, where P values of P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered significant.Asterisks were used for comparisons between NTC and siPOLA1, triangles for comparisons between NTC and mock, hash symbols for comparisons between mock and siPOLA1.